A method for the isolation of homogeneous preparation of CNS myelin proteolipid protein has been developed in this laboratory. Studies are in progress to determine the chemical and immunological properties of myelin PLP. Using the isolated homogeneous preparation of rat brain myelin PLP, we are currently engaged in determining the N-terminal amino acid sequence by direct Edman degradation of the native, reduced or oxidized protein. Cyanogen bromide fragments and tryptic peptide maps of PLP will be prepared and the amino acid sequence of these polypeptides will be deduced by Edman degradation by the use of an automatic amino acid sequencer. Similarly the complete amino acid sequence of human brain myelin PLP will be determined. By making use of the same preparations of rat brain myelin PLP, we have successfully produced precipitating antibodies in both the rabbit and goat. Immunofluorescence localization of PLP demonstrated bright specific fluorescence limited to the myelin sheath of axons in all areas of the normal adult rat brain examined. The availability of antisera to PLP will enable us to determine by immunofluorescence the changes in the protein in normal areas and in plaques of brain tissue from patients afflicted with multiple sclerosis. In addition, we will study the appearance of myelin specific proteolipid protein in developing rat brain immunohistochemically with light and electron microscopes. Such an approach will enable us to determine where PLP is localized in CNS myelin, whether it is present within the interfascicular oligodendrocytes, and when it first becomes apparent in these respective structures. Similarly, we will test the presence of myelin PLP in cultured cell lines cloned from astrocytoma, oligodendroglioma and neuroblastoma, and indirect approach to the study of pure preparations of various cell types in the CNS. Since rabbit antiserum forms precipitating antibodies, we will attempt to standardize a solid phase radioimmunoassay for PLP. The development of such a technique for a membrane specific protein like PLP will permit us to determine the concentration of PLP in the nanogram range in the blood and cerebrospinal fluid and in normal and plaque areas of brain tissue from multiple sclerosis patients.